Infection with the bacterium Clostridium difficile causes symptoms ranging from mild to severe diarrhoea with life-threatening complications and remains a significant burden to healthcare systems throughout the developed world. Two potent cytotoxins, TcdA and TcdB are the prime mediators of the syndrome and rapid neutralisation of these would afford significant benefits in disease management. In the present study, a broad range of non-toxic, recombinant fragments derived from TcdA and TcdB were designed for soluble expression in E. coli and assessed for their capacity to generate a potent toxin-neutralising immune response as assessed by cell-based assays. Significant differences between the efficacies of isolated TcdA and TcdB regions with respect to inducing a neutralising immune response were observed. While the C-terminal repeat regions played the principal role in generating neutralising antibodies to TcdA, in the case of TcdB, the central region domains dominated the neutralising immune response. For both TcdA and TcdB, fragments which comprised domains from both the central and C-terminal repeat region of the toxins were found to induce the most potent neutralising immune responses. Generated antibodies neutralised toxins produced by a range of C. difficile isolates including ribotype 027 and 078 strains. Passive immunisation of hamsters with a combination of antibodies to TcdA and TcdB fragments afforded complete protection from severe CDI induced by a challenge of bacterial spores. The results of the study are discussed with respect to the development of a cost effective immunotherapeutic approach for the management of C. difficile infection.